A peer-reviewed open-access journal ZooKeys 857: 1–15 (2019) Redescription of Syncarpa composita 1 doi: 10.3897/zookeys.857.32654 RESEARCH ARTICLE http://zookeys.pensoft.net Launched to accelerate biodiversity research A redescription of Syncarpa composita (Ascidiacea, Stolidobranchia) with an inference of its phylogenetic position within Styelidae Naohiro Hasegawa1, Hiroshi Kajihara2 1 Department of Natural History Sciences, Graduate School of Science, Hokkaido University, Kita 10 Nishi 8 Kitaku, Sapporo, Hokkaido 060-0810, Japan 2 Faculty of Science, Hokkaido University, Kita 10 Nishi 8 Kitaku, Sapporo, Hokkaido 060-0810, Japan Corresponding author: Naohiro Hasegawa ([email protected]) Academic editor: Tito Lotufo | Received 25 December 2018 | Accepted 6 May 2019 | Published 24 June 2019 http://zoobank.org/2183A9EC-C4B7-4863-B03B-EB5346D7B95E Citation: Hasegawa N, Kajihara H (2019) A redescription of Syncarpa composita (Ascidiacea, Stolidobranchia) with an inference of its phylogenetic position within Styelidae. ZooKeys 857: 1–15. https://doi.org/10.3897/ zookeys.857.32654 Abstract Two species of styelid colonial ascidians in the genus Syncarpa Redikorzev, 1913 are known from the northwest Pacific. The valid status of the lesser known species, Syncarpa composita (Tokioka, 1951) (type locality: Akkeshi, Japan), is assessed here. To assess the taxonomic identity of S. composita, we com- pared one of the syntypes and freshly collected topotypes of S. composita with a syntype of S. oviformis Redikorzev, 1913 (type locality: Ul’banskij Bay, Russia). Specimens of S. composita consistently differed from the syntype of S. oviformis in the number of oral tentacles, the number of size-classes of transverse vessels, and the number of anal lobes. In this paper, S. composita is redescribed as distinct from S. oviformis, and its phylogenetic position inferred within Styelidae based on the 18S rRNA and cytochrome c oxidase subunit I gene sequences. In our phylogenetic tree, Syncarpa formed a well-supported clade together with Dendrodoa MacLeay, 1824. In Syncarpa and Dendrodoa, a single gonad is situated on the right side of the body, which is unique among Styelidae, and thus can be a synapomorphy for this clade. Keywords Chordata, COI, phylogeny, Sea of Okhotsk, taxonomy, Urochordata Copyright N. Hasegawa, H. Kajihara. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Naohiro Hasegawa & Hiroshi Kajihara / ZooKeys 857: 1–15 (2019) Introduction Syncarpa Redikorzev, 1913 is a member of the ascidian family Styelidae and consists of two species, Syncarpa composita (Tokioka, 1951) and S. oviformis Redikorzev, 1913. The two nominal species S. corticiformis Beniaminson, 1975 and S. longicaudata Skalkin, 1957, all from the Northwest Pacific, have been synonymized withS. oviformis by Sanamyan (2000). This genus is defined by the following four characters: i) colonial, with zooids reproducing asexually, ii) a single, well-developed fold is present on each side of the pharynx, iii) a single gonad is situated on the right side of the body, and iv) the gonad has several branches. Syncarpa composita is only known by the original description based on material from Akkeshi, Japan (Tokioka 1951). It was originally placed in a new monotypic genus Syndendrodoa Tokioka, 1951, which has been syn- onymized with Syncarpa by Nishikawa (1995). The phylogeny of ascidians including styelids has been investigated by Zeng et al. (2006), Pérez-Portela et al. (2009), Tsagkogeorga et al. (2009), Alié et al. (2018), and Delsuc et al. (2018). Among these, Alié et al.’s (2018) analysis was based on 4908 genes and included 16 OTUs from Styelidae. It recovered Styelidae as monophyletic with maximum branch-support values, which turned out to be sister to part of paraphyletic Pyuridae. Alié et al.’s (2018) phylogeny showed three major clades for Styelidae: i) Polyzoinae + Botryllinae, ii) Dendrodoa + Polycarpa + Polyandrocarpa zorritensis (Van Name, 1931), and iii) Astrocarpa + Styela. However, no member of Syncarpa has ever been placed on a phylogenetic context in any of the previous studies. The aims of this study are to assess the taxonomic identity of S. composita based on type specimens and freshly collected topotypes andto infer the species’ phylogenetic position among Styelidae. In this paper, we redescribe the species and present the re- sults of a multi-gene molecular analysis. Materials and methods Eleven topotype colonies of S. composita were freshly collected by dredging, snorke- ling, and SCUBA diving in the type locality, Akkeshi Bay, at depths of 3–5 m in June, August, and September 2017, and July 2018 (Table 1). One of the colonies was pho- tographed underwater and in the laboratory with a Nikon COOLPIX AW130 digital camera. The live colonies were anesthetized with menthol; then a part of a zooid was cut off along with the tunic from each colony and preserved in 99% EtOH for DNA extraction. The colonies were preserved in 10% formalin-seawater for morphological observation; zooids were removed from the colonies and then dissected for morpho- logical examination. Larvae for histological observation were dehydrated in an etha- nol series, cleared in xylene, embedded in paraffin wax, sectioned at 5 µm thickness, and stained with hematoxylin and eosin. After sections were mounted on glass slides in Entellan New (Merck, Germany), they were observed under an Olympus BX51 Redescription of Syncarpa composita 3 Table 1. List of specimens newly collected in this study with species, family, sampling date, sampling site, GenBank accession numbers for 18S and COI sequences included in the analysis, and catalog numbers. Family Species Sampling date Sampling site GenBank accession number Catalog number 18S COI Botrylloides violaceus 30 Mar 2017 Oshoro Bay LC432326 LC432331 ICHUM 5826 Styela clava 26 Aug 2017 Shukutsu LC432329 LC432334 ICHUM 5827 Styela plicata 10 Jul 2017 Moroiso Bay LC432328 LC432333 ICHUM 5828 25 Jun 2017 – – ICHUM 5815 25 Jun 2017 – – ICHUM 5816 2 Aug 2017 LC432325 LC432330 ICHUM 5817 7 Sep 2017 – – ICHUM 5818 Styelidae 7 Sep 2017 – – ICHUM 5819 Syncarpa composita 7 Sep 2017 Akkeshi Bay – – ICHUM 5820 7 Sep 2017 – – ICHUM 5821 7 Sep 2017 – – ICHUM 5822 7 Sep 2017 – – ICHUM 5823 13 Jul 2018 – – ICHUM 5824 13 Jul 2018 – – ICHUM 5825 Pyuridae Pyura mirabilis 21 Jun 2017 Oshoro Bay LC432327 LC432332 ICHUM 5829 compound microscope and photographed with a Nikon D5200 digital camera. These voucher specimens have been deposited in the Invertebrate Collection of the Hok- kaido University Museum (ICHUM), Sapporo, Japan. For comparison, specimens deposited in the Seto Marine Biological Laboratory (SMBL), Shirahama, Japan, and the Zoological Institute of the Russian Academy of Sciences (ZIRAS), St. Petersburg, Russia, were also examined. Total genomic DNA was extracted from a piece of the body wall tissue for eight specimens of S. composita as well as one specimen each of Botrylloides violaceus Oka, 1927, Pyura mirabilis (Drasche, 1884), Styela clava Herdman, 1881, and Styela plicata (Lesueur, 1823) (Table 1). The tissue was placed in a 1.5 mL tube after air-dried, then mixed with 180 µL of ATL buffer (Qiagen, Hilden, Germany) and 20 µL of proteinase K (>700 U/mL, Kanto Chemical, Tokyo, Japan), and incubated at 55 °C for ca. 10 h. To the lysis solution, 200 µL of AL buffer (Qiagen) was added and incubated at 70 °C for 10 min; then 210 µL of 99% EtOH was added. The rest of the DNA extraction was carried out following Boom et al.’s (1990) silica method. Two gene markers were amplified from the genomic DNA by PCR. The nuclear 18S rRNA (18S) gene was amplified with the primer pair 1F/9R (Giribet et al. 1996). The mitochondrial cytochromec oxidase subunit I (COI) gene was amplified with the primers Sty_COI_F2 (5'-TTTGCCTTTAATAGTAAGAAGTCC-3') and Sty_COI_ R1 (5'-CATCAAAACAGATGCTGATA-3') for S. composita and with the primer pair LCO1490/HCO2198 (Folmer et al. 1994) for the other ascidians. PCRs were per- formed in a 10-µL total reaction volume with 3 µL of each primer pair (10 µM), 0.5 µL of TaKaRa Ex Ta q (TaKaRa, Kusatsu, Japan), 10 µL of 10 × Ex Ta q Buffer (TaKaRa), 8 µL of dNTP mixture (TaKaRa), 1 µL of extracted DNA, and 68.5 µL of deionized water. Thermal cycling condition was 94 °C for 2 min; 35 cycles of 94 °C for 4 Naohiro Hasegawa & Hiroshi Kajihara / ZooKeys 857: 1–15 (2019) 45 sec, 52 °C for 90 sec (for 18S) or 55 °C for 50 sec (for COI), and 72 °C for 55 sec; then 72 °C for 5 min. Amplification was verified by electrophoresis in 1% agarose gel. The PCR products were purified through enzymatic reaction with 24 mU/µL of Exonuclease I (TaKaRa) and 4.9 mU/µL of Shrimp Alkaline Phosphatase (TaKaRa). The purified PCR products were sequenced directly with a BigDye Terminator ver. 3.1 Cycle Sequence Kit (Applied Biosystem, Foster, CA, USA) and 3730 Genetic Analyzer (Applied Biosystems), using the same primer pairs for amplification, as well as the fol- lowing internal primers for 18S: 3F, 5R (Giribet et al. 1996); and 2, bi (Whiting et al. 1997). Base calling was performed with GeneStudio Professional Edition ver. 2.2.0.0 (GeneStudio, Suwanee, GA, USA). To infer the phylogenetic position of S. composita, 18S and COI sequences of 24 species of Styelidae were obtained from GenBank (Table 2). For 18S, alignment was car- ried out by MAFFT ver. 7 using the E-INS-i strategy (Katoh and Standley 2013); am- biguous sites were removed by using Gblocks ver. 0.91b (Castresana 2002). For COI, nucleotide sequences were manually edited by MEGA ver. 5.2.2 (Tamura et al. 2011) so that translated amino acid sequences were aligned straightforward without indels.